Methods for the detection, treatment, and prevention of neurodegeneration

ABSTRACT

In general, the invention provides methods for identifying genes involved in neurodegeneration and therapeutics for treating animals with a neurodegenerative disease. Methods and kits for the detection of compounds which enhance neuroprotection and diagnostic kits for the detection of neurodegenerative diseases are also a part of the invention.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] This invention was made in part with support from the Federal government through NIH Grant No. 1RO1NS32196-04. The Federal government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] The invention relates to methods and reagents for diagnosing, treating, and preventing neurodegeneration.

[0003] Loss of neurons by a degenerative process is a major pathological feature of many human neurological disorders. Neuronal cell death can occur as a result of a variety of conditions including traumatic injury, ischemia, neurodegenerative diseases (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), stroke, or trauma), or as a normal part of tissue development and maintenance. Several inherited disorders produce late onset neuron loss, each of which is highly specific for particular neural cell types. Nine genes have been cloned that are associated with susceptibility to these various neurological disorders (e.g., Huntington's disease, ataxin, and ALS); however, only in the case of Kennedy's syndrome is the biochemical function of the affected gene, the androgen receptor, understood (La Spada et al., Nature 352: 77-79, 1991). Epileptic seizures and stroke also produce neurodegeneration in humans and rodents.

SUMMARY OF THE INVENTION

[0004] In general, the invention features methods for the detection, treatment, and prevention of disorders involving neurodegeneration.

[0005] In a first aspect, the invention features a method for identifying a compound to treat or prevent the onset of a neurodegenerative disorder. The method involves contacting a cell that includes a reporter gene operably linked to a cAMP regulatory gene or promoter with a candidate compound and measuring the expression of the reporter gene, where a change in reporter gene expression in response to the candidate compound identifies a compound that is useful to treat or prevent the onset of a neurodegenerative disorder.

[0006] In various preferred embodiments of the first aspect of the invention, the cAMP regulatory gene may be an acy-1 gene, an eat-4 gene, an unc-36 gene, or a glutamate receptor-encoding gene. In another preferred embodiment, the change in reporter gene expression is a decrease in expression.

[0007] In a second aspect, the invention features a cell for identifying a compound to treat or prevent the onset of a neurodegenerative disorder that includes a reporter gene operably linked to a cAMP regulatory gene or promoter.

[0008] In various embodiments of the above aspects, the cell is present in an animal, which may be a nematode (e.g., C. elegans) or a mammal (e.g., a rodent).

[0009] In a third aspect, the invention features a method for treating or preventing the onset of a neurodegenerative disorder in a mammal that includes administering to the mammal a therapeutically effective amount of a compound that decreases a neuronal cAMP level. In a preferred embodiment of this aspect of the invention, the mammal is a human.

[0010] In a fourth aspect, the invention features a method for identifying a mammal (for example, a human) having or likely to develop a neurodegenerative disorder which includes determining whether the mammal has an increased level of cellular cAMP in a neuron, where an increased level indicates that the mammal has or is likely to develop a neurodegenerative disorder.

[0011] In a fifth aspect, the invention features a method for identifying a mammal (for example, a human) having or likely to develop a neurodegenerative disorder which involves determining whether the mammal has a mutation in a cAMP regulatory gene. In various preferred embodiments of this aspect, the mutation is in an adenylyl cyclase gene (e.g., the acy-1 gene), or in an unc-36 or eat-4 gene. In other preferred embodiments, the mutation is in a gene encoding a Gα_(s) subunit; and the mutation results in an increase in a neuronal cAMP level.

[0012] In a preferred embodiment of various aspects of the invention, the neurodegenerative disorder is Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, stroke, or epilepsy.

[0013] In a sixth aspect, the invention features a method for identifying a gene involved in neurodegeneration that involves providing a nematode (for example, C. elegans) that includes an expression construct that includes a promoter derived from a cAMP regulatory gene operably linked to a reporter gene, isolating a mutant of the nematode exhibiting an altered level of reporter gene expression, and identifying the gene comprising the mutation, wherein the gene is involved in neurodegeneration.

[0014] In a seventh aspect, the invention features a method for identifying a gene involved in neurodegeneration that involves providing a nematode (for example, C. elegans) that includes a glutamate receptor (GluR) promoter operably linked to a gene encoding a GTP-ase defective Gα_(s) subunit, isolating a mutant of the nematode exhibiting a decreased level of paralysis and neurodegeneration, and identifying the gene that includes the mutation, wherein the gene is involved in neurodegeneration.

[0015] In an eighth aspect, the invention features a mammalian (for example, a human) EAT-4 polypeptide, and a vector and cell containing the nucleic acid.

[0016] In a final aspect, the invention provides a method for identifying a gene involved in neurodegeneration involving the steps of a) providing a cell that includes a cAMP regulatory gene promoter operably linked to a reporter gene; b) introducing into the cell a candidate gene capable of expressing a candidate protein; and c) measuring reporter gene expression in the cell, where an increase in reporter gene expression in the presence of the candidate protein indicates that the candidate gene is involved in neurodegeneration.

[0017] In preferred embodiments, the cell is yeast; and the cAMP regulatory gene is an acy-1 gene, an eat-4 gene, an unc-36 gene, or a glutamate receptor-encoding gene.

[0018] As used herein, by “protein” or “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0019] By “neurodegenerative disorder” is meant a disorder which is characterized by the death or loss of function of neuronal cells, also known as neurons. Neuronal death or loss of function can be associated with a number of diseases and syndromes including, without limitation, stroke, epilepsy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease.

[0020] By “Gα_(s)-induced toxicity” is meant the neurodegeneration resulting from expression of the GTP-ase defective Gα_(s) protein.

[0021] By “reporter gene” is meant any gene which encodes a product whose expression is detectable. A reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ), toxicity (e.g., HER-1), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labelled antibody).

[0022] By “cAMP regulatory gene” is meant any gene whose product regulates or is regulated by cAMP. Exemplary gene products include ACY-1, UNC-36, and EAT-4. Other preferred cAMP regulatory gene products include the ionotropic (cation) glutamate receptors (iGluRs), the Cl ionotropic glutamate receptors (GluCls), and the metabotropic glutamate receptors (mGluRs).

[0023] By “operably linked” is meant that a gene and a regulatory sequence are connected in such a way as to permit expression of the gene product under the control of the regulatory sequence.

[0024] By “purified nucleic acid” is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autosomally replicating plasmid or virus; or into the genomic DNA or a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0025] By a “transgene” is meant a nucleic acid sequence which is inserted by artifice into a cell and becomes a part of the genome of that cell and its progeny. Such a transgene may be partly or entirely heterologous to the cell.

[0026] By “mammalian eat-4 polypeptide or mammalian EAT-4” is meant an amino acid sequence derived from a mammalian cell which shares at least 50%, preferably 70%, more preferably 80%, and most preferably 90% amino acid sequence identity with a C. elegans eat-4 amino acid sequence (SEQ ID NO: 1). Preferably, such a polypeptide is capable of at least partially complementing a C. elegans eat-4 mutation.

[0027] By “acy-1 polypeptide or ACY-1” is meant an amino acid sequence which is substantially identical to the amino acid sequence provided in FIG. 5 (SEQ ID NO: 2).

[0028] By “substantially identical” is meant an amino acid sequence or nucleic acid sequence which shares identity with another of the same class. Preferably, such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical to the sequence described in the references provided herein. For polypeptides, the length of comparison sequences will generally be at least 15 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acids, the length of comparison sequences will be at least 45 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 105 nucleotides. Identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of identity to various substitutions, deletions, substitutions, and other modifications.

[0029] Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIGS. 1A and 1B are photographs of neuronal cells from young Caenorhabditis elegans larvae co-expressing green fluorescent protein (GFP) with GTP-ase defective rat Gα_(s) as seen morphologically (FIG. 1A), as well as in bright field optics (FIG. 1B).

[0031]FIG. 2 is a table listing the extent of swelling and cytotoxicity of PVC neurons resulting from the expression of the α_(s)(gf) transgene in various genetic backgrounds. Statistical differences between genotypes were determined by the method of attributable risk described in J. Devore, Probability and statistics for engineering and the sciences (Brooks/Cole, Belmont, ed. second, 1987). Multiple comparisons were compensated for by setting p<0.005 as the threshold for significance.

[0032]FIG. 3 is an amino acid sequence of the EAT-4 protein (SEQ ID NO: 1).

[0033]FIG. 4 is a schematic diagram showing the genetic and physical map position of the acy-1 gene on the F17C8 cosmid.

[0034]FIG. 5 is a set of schematic diagrams of the predicted structures of the acy-1 gene and the GFP fusion protein (KP#107). Positions of the acy-1 mutations nu327, nu343, and nu329 are indicated.

[0035]FIG. 6 is an amino acid sequence of the ACY-1 protein (SEQ ID NO: 2). The ACY-1 sequence (top) is shown aligned with the mouse adenylyl cyclase type 9 amino acid sequence (bottom). Underlined sequences indicate predicted transmembrane domains. Positions of the acy-1 mutations nu327, nu343, and nu329 are indicated.

[0036]FIGS. 7A and 7B are photographs of GFP-expressing PVC neurons in adult α_(s)(gf) (FIG. 7A) and adult α_(s)(gf);acy-1(nu343) (FIG. 7B) C. elegans.

[0037]FIGS. 8A and 8B are photographs illustrating KP#107 acy-1::gfp fusion gene expression in neurons (FIG. 8A) and muscle (FIG. 8B).

[0038]FIGS. 9A and 9B are photographs of PVC neurons from unc-18 L1 larvae as seen with bright field (FIG. 9A) and fluorescence (FIG. 9B) optics.

[0039]FIGS. 10A and 10B are photographs of PVC neurons from unc-18 adults as seen with bright field (FIG. 10A) and fluorescence (FIG. 10B) optics.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The invention described herein is based upon genetic studies of the nematode, Caenorhabditis elegans. Constitutive activation of the GTP-binding protein Gα_(s) was found to induce neurodegeneration. A screen for mutations that blocked Gα_(s)-induced killing identified a gene, acy-1, which encodes a protein that is highly similar (40% identical) to mammalian adenylyl cyclases, indicating that Gα_(s)-induced neurotoxicity is likely mediated by changes in cyclic adenosine monophosphate (cAMP) levels. This discovery enables methods and reagents for diagnosing and treating neurodegeneration.

[0041] Gα_(s)-induced Neurotoxicity

[0042] Although neurodegeneration is a major feature in a variety of human neurological disorders, relatively little is known about the signal transduction pathways that lead to neurotoxicity, nor how these pathways could be manipulated to protect against neuron loss in these diseases. Two critical questions in the pathogenesis of human neurodegenerative disorders are (1) what factors predispose particular neurons to undergo degeneration and (2) what is the biochemical mechanism of degeneration. A genetic model for excitotoxicity in the nematode Caenorhabditis elegans was developed to address these questions.

[0043] In particular, a rat cDNA encoding a GTPase-defective (Q227L) Gα_(s) subunit, hereafter referred to as α_(s)(gf), was expressed in C. elegans neurons using the glr-1 glutamate receptor (GluR) promoter. The expression vector, KP#20, was constructed by inserting into a derivative of the C. elegans glr-1 expression vector CX#1 (as described in Chalfie et al., Science 263: 802-805, 1994), a 1.5 kb NcoI-XhoI fragment encoding a GTPase defective (Q227L, KP#20) mutant rat Gα_(s) cDNA. C. elegans transgenic for α_(s)(gf) were prepared by microinjecting the KP#20 expression construct together with a glr-1::gfp plasmid (the KP#6 vector) using lin-15 (Huang et al., Mol. Biol. Cell. 5, 395-412, 1994) as a transformation marker. A stable line carrying glr-1 expression constructs for both GFP and the GTPase defective Gα_(s)(nuIs5) was isolated following 3500 rads of γ-irradiation. The glr-1 promoter was chosen because it is highly expressed, and because glr-1-expressing cells control locomotion, an easily assayed behavior. The glr-1 promoter is expressed in 17 classes of neurons, including the interneurons (AVB, PVD, AVA, and AVD) required for locomotion. The glr-1 expressing neurons are as follows: AVG, AVJ, DVC, PVC, PVQ, RIG, RIS, RMD, RMEL/R, SMD, URY, as well as the six ASH synaptic targets AIB, AVA, AVB, AVD, AVE, and RIM (Hart et al., Nature 378: 82-85, 1995; Maricq et al., Nature 378: 78-81, 1995).

[0044] Since Gα_(s) was co-expressed with the green fluorescent protein (GFP) of Aequorea (Chalfie et al., supra), examination of the morphology of Gα_(s)-expressing cells was possible. Transgenic glr-1::α_(s)(gf) animals were found to be paralyzed. As shown in FIGS. 1A and 1B, a subset of the Gα_(s)-expressing neurons in young larvae swelled to several times their normal diameter. The swelling was apparent by the morphology of GFP expressing cells (FIG. 1A) and by their appearance in bright field optics (FIG. 1B) as enlarged, apparently vacuolated cells often with an intact nucleus. The interneurons AVE and AVD were swollen compared to neighboring unaffected cells which have been marked in FIGS. 1A and 1B with asterisks. 88% of the PVC neurons swelled, 5% of RIG neurons swelled, and none of the URY cells swelled in first stage (L1) glr-1::α_(s)(gf) larvae. The neurotoxicity occurred in two phases; subsequent to swelling, the swollen cells eventually disappeared, presumably because the cells had died. In glr-1::α_(s)(gf) animals, 89% of the PVC neurons degenerated, as summarized in the table in FIG. 2. Other glr-1 expressing cells degenerated at lower frequencies, including AVA, AVD, AVE, AVG, PVQ, RIG, and SMD. Expression of a constitutively active rat Gα_(s) cDNA was found to cause neurotoxicity in C. elegans. Characterization of the neurodegenerative phenotype in the resulting glr-1::α_(s)(gf) was made as follows: Swollen or missing cells were identified by examining the morphology of GFP-expressing cells. Gα_(s)-induced neurotoxicity in various genetic backgrounds was quantitated as the number of swollen PVC neurons in L1 larvae, and the percentage of PVC neurons that were missing or swollen in adults hermaphrodites. These results suggested that exaggerated Gα_(s) signaling killed neurons.

[0045] The phenotype of Gα_(s)-induced neurotoxicity was identical to the neurotoxicity due to excessive signaling by the excitatory neurotransmitter glutamate, which has been termed excitotoxicity. Excitotoxic neuron loss occurs in two phases. First, acute neuron loss is associated with swelling of cell bodies and is dependent on extracellular ionic conditions. Cell swelling is the consequence of depolarization of membrane potential by excitotoxic agonists, which leads to the influx of Na⁺ and Cl⁻ ions, and water (Olney, Adv. Exp. Med. Biol. 203: 631-645, 1986; Choi, J. Neurosci. 7: 369-379, 1987; Choi, Neuron 1: 623-634, 1988). Second, delayed neuron loss in excitotoxicity is not dependent on the extracellular ionic conditions, but is correlated with elevations of intracellular Ca²⁺ and chronic activation of immediate early genes (e.g., fos and jun) (Smeyne et al., Nature 363: 166-169, 1993. Hence, Gα_(s)-induced neurotoxicity is most likely excitotoxicity.

[0046] Neurons Differed Greatly in their Susceptibility to Gα_(s)-induced Toxicity

[0047] The mec-7 gene product, MEC-7 tubulin, is abundantly expressed in 5 neurons, called touch cells, that sense light touch to the worm's body (Savage et al., Genes Dev. 3: 870-81, 1989; Hamelin et al., EMBO 11: 2885-2893, 1992; Mitani et al., Development 119: 773-783, 1993). To further investigate the specificity of Gα_(s)-induced toxicity, α_(s)(gf) was expressed in C. elegans utilizing the mec-7 promoter. The mec-7::α_(s)(gf) expression plasmid (KP#7) was constructed by ligating the 1.5 kb NcoI-XhoI Gα_(s)(Q227L) into the mec-7 expression vector pPD52.102. C. elegans transgenic for the mec-7::α_(s)(gf) expression plasmid were prepared by microinjecting the KP#7 expression construct together with a mec7::gfp plasmid using lin-15 (Huang et al., supra) as a transformation marker. A stable line carrying mec-7 expression constructs for both GFP and the GTPase defective Gα_(s)(nuIs5) was isolated following γ-irradiation.

[0048]C. elegans expressing the mec-7::α_(s)(gf) transgene were found to be indistinguishable from wild type animals, having no obvious defect in touch sensitivity nor in the morphology of the touch cells. Hence, the effects of Gα_(s) on neural activity and on neurotoxicity were cell type specific.

[0049] Mutations that Blocked Gα_(s)-induced Neurotoxicity

[0050] Both the glr-1 and the mec-7 expression constructs supported the notion that the effects of Gα_(s) on neural activity and on neurotoxicity were cell type specific. Since the mec-7 promoter is very highly expressed in the touch neurons (Savage et al., supra; Hamelin et al., supra; Mitani et al., supra), the results also suggested that the cell type specificity could not be overcome by high levels of Gα_(s) expression. To identify the targets of Gα_(s), mutations that block Gα_(s)-induced paralysis and neurotoxicity were isolated by identifying mutations isolated from the F2 self-progeny of EMS mutagenized (5 μl/ml) hermaphrodites that restored normal locomotion rates to α_(s)(gf) homozygotes. Candidate suppressor mutants (7500 hapliod genomes) were subsequently screened for reduction of Gα_(s)-induced swelling in L1 larvae which led to the isolation of 3 semidominant mutations which blocked Gα_(s)-induced paralysis and neurotoxicity

[0051] Mutations in acy-1 Blocked Gα_(s)-induced Neurotoxicity

[0052] In two factor mapping experiments, the three mutations that blocked Gα_(s)-induced neurotoxicity were all found to be linked to dpy-17. Three factor mapping placed these mutations between emb-5 and dpy-17: (nu327 dpy-17) 37/37 unc-32; (nu329 dpy-17) 16/16 unc-32; (nu343 dpy-17) 4/4 unc-32; unc-79 (6/14) MJ#NEC2 (5/14) nu329 (3/14) dpy-17; emb-5 (1/16) nu327 (15/16) dpy-17. As illustrated in the schematic diagram of FIG. 4, two of the three mutations were mapped to a 1.5 cM genetic interval between MJ#NEC2 and dpy-17 on the F17C8 cosmid. The cosmid was then microinjected into acy-1(nu327); nuIs5 animals, and transgenic lines were isolated using goa-1::gfp (KP#13) (Segalat, et al., Science 267, 1648-1651, 1995) as a transformation marker. Four independent lines carrying a cosmid from this interval (F17C8) were obtained, two of which corrected the mutant phenotype of acy-1(nu327) animals, i.e., they had increased degeneration of the PVC neurons. This is shown on Table 1. TABLE 1 Transgenes containing the F17C8 cosmid rescue the acy-1(nu327) mutant phenotype genotype % PVC degeneration acy-1(nu327);a_(s)(gf) 12 acy-1(nu327);a_(s)(gf); nuEX(F17C8) 75 a_(s)(gf) 88

[0053] In addition, FIG. 5 shows that all three alleles corresponded to mutations in the predicted exons of the gene F17C8.1, one of two predicted adenylyl cyclase genes in the C. elegans genome database. This adenylyl cyclase gene has been named acy-1. Furthermore, FIG. 6 shows the results of a Genbank database scan for sequences related to acy-1 (SEQ ID NO: 2). The amino acid sequence of ACY-1 was found to be 40% identical at the amino acid level to mouse adenylyl cylase type 9. It is unclear why the acy-1 mutations were partially dominant. Analysis of the molecular nature of the mutations suggested that they were simple loss of function mutations. For example, nu329 and nu343 were predicted to disrupt pre-mRNA splicing. Indeed, as is shown in FIGS. 7A and 7B, the GFP-expressing PVC neurons which were typically missing in α_(s)(gf) adult transgenic worms (FIG. 7A) were present in α_(s)(gf);acy-1(nu343) (FIG. 7B). Thus, it is possible that α_(s)(gf) animals were highly sensitive to changes in cAMP levels. Overall, the results suggested that Gα_(s) neurotoxicity was mediated by changes in intracellular cAMP.

[0054] Physiological Function of ACY-1

[0055] To determine the physiological function of ACY-1, an analysis of acy-1 expression was carried out. A deleted derivative (KP#106) of the cosmid F17C8 was isolated by digesting with AflII and re-ligating. KP#106 contained the entire 8.35 kb acy-1 genomic region together with the 5.2 kb 5′ and 4.9 kb 3′ flanking sequences. An acy-1::gfp expression vector (KP#107) was constructed by PCR amplification of a 1.7 kb fragment containing the GFP coding region and the unc-54 transcription terminator from pPD95.75, followed by ligation of this fragment into the unique Asp718 site in KP#106, creating a fusion protein containing the first 6 exons of acy-1 fused to GFP. The ACY-1::GFP fusion protein contained 6 predicted transmembrane domains of ACY-1, and was therefore membrane localized. Transgenic animals carrying KP#107 were isolated by microinjection using lin-15 (Huang et al., supra) as a transformation marker. Expressing cells were identified based on their morphology and nuclear positions.

[0056] The expression pattern of acy-1 was determined by analyzing the GFP reporter construct. As is shown in FIGS. 8A and 8B, the acy-1::gfp fusion protein was expressed in virtually all neurons (FIG. 8A) and body muscles (FIG. 8B). In FIG. 8A, ACY-1 expression in the two ventral rows of body muscles (arrows) and in the ventral cord neurons and neuropile (lines) is shown. In FIG. 8B, expression of ACY-1 in the vulva muscles (arrow heads) is shown. Nearly all of the 302 neurons in adult C. elegans appeared to express ACY-1. Cell bodies were identified based upon the bright fluorescence in the intracellular membranes (which are presumably the endoplasmic reticulum of Golgi apparatus). ACY-1 did not appear to be expressed in non-neural tissues or in the pharynx. These results indicated that the ACY-1 adenylyl cyclase is likely to participate in many neural signaling pathways. Therefore, we expected that acy-1 mutants would have defects in behavior or development. Consistent with this notion is that mutations which inactivated the C. elegans Gα_(s) subunit (GSA-1) were found to be homozygous lethal. Surprisingly, we observed that acy-1 homozygotes were nearly indistinguishable from wild type animals. This result suggested that the essential function of GSA-1 was mediated by some other adenylyl cyclase. Alternatively, acy-1 and other adenylyl cyclases could act redundantly in the essential GSA-1 pathways.

[0057] Activated Gα_(s) Induced Neurotoxicity by Excitotoxicity

[0058] Several previously identified genes were considered good candidates for mediating the toxic effects of Gα_(s). Two cyclic nucleotide gated ion channel (CNGC) subunit genes tax-2 and tax-4 (Coburn and Bargmann, Neuron 17: 695-706, 1996; Komatsu et al., Neuron 17: 707-718, 1996) are not expressed in glr-1 expressing cells and hence are unlikely targets. The mec-6, unc-8, and deg-1 genes have been previously implicated in neurodegeneration (Chalfie and Wolinksy, Nature 345: 410-416 (1990); Driscoll and Chalfie, Nature 349: 588-593 ,1991; Shreffler et al., Genetics 139: 1261-1272, 1995; Tavernarakis et al., Neuron 18: 107-119, 1997), and the DEG-1 and UNC-8 proteins are similar to mammalian epithelial sodium channel subunits (ENaC), which are potently activated by cAMP-dependent protein kinase (PKA) (Sariban-Sohraby et al., J. Biol. Chem. 263: 13875-13879, 1988; Oh et al., Am. J. Physiol. 265: C85-C91, 1993; Bubien et al., J. Biol. Chem. 269: 17780-17783, 1994). The unc-2, unc-36, and egl-19 genes encode subunits of voltage-dependent Ca²⁺-channels (Schafer and Kenyon, Nature 375: 73-78, 1995) which are likely to be regulated by PKA (Curtis and Catterall, Proc. Natl. Acad. Sci. USA 82: 2528-2532, 1985) and have also been implicated in neurodegeneration. The glr-1 gene encodes an ionotropic GluR (Hart et al., supra; Maricq et al., supra). GluRs have been implicated in neurotoxicity in mammals (Olney, Adv. Exp. Med. Biol. 203, 631-645, 1986; Choi, J., Neurosci. 7: 369-379, 1987; and Choi, Neuron 1, 623-634, 1988), and PKA augments the response of mammalian neurons to glutamatergic agonists (Greengard et al., Science 253: 1135-1138, 1991).

[0059] To examine the above genes for a possible role in Gα_(s) induced toxicity, the neurodegenerative phenotype was characterized as described above. As shown in FIG. 2, of the candidate genes, only the unc-36 mutation significantly reduced Gα_(s)-induced cytotoxicity. Interestingly, the unc-36 mutation had no effect on cell swelling. Since UNC-36 Ca²⁺ channels were required for cytotoxicity, these results suggested that Gα_(s) cytotoxicity was mediated in part by either Ca²+ influx or depolarization of the affected cells. All other candidate genes had no effect on either neuron swelling or deaths in glr-1::α_(s)(gf) animals. Our results do not exclude the possibility that these other candidate PKA targets also play a role in Gα_(s)-induced toxicity. For example, more than one type of channel may be capable of mediating the toxic effects of Gα_(s), in which case neurotoxicity would be prevented only in multiply mutant animals.

[0060] The glr-1 Mutation was Unlikely to Completely Abolish Glutamate Signaling in vivo

[0061] Given its role in excitotoxicity in mammals, the requirement of endogenous glutamate signaling for Gα_(s) neurotoxicity was tested. Although the glr-1 mutation was not neuroprotective, it was possible that cAMP toxicity was mediated by exaggerated responses to endogenous glutamate. The C. elegans genome sequence (currently ˜-70% complete) predicted six additional ionotropic GluR subunits; therefore, the glr-1 mutation was unlikely to completely abolish glutamate signaling in vivo.

[0062] Eat-4 Mutant Alleles Eliminated ASH-mediated Touch Sensitivity

[0063] Prior work had shown that ASH sensory neurons mediated an aversive response to three distinct stimuli (nose touch, osmotic shock, and volatile repellents), and that the ASH-mediated touch response required functional GLR-1 glutamate receptors in synaptic targets of ASH (Hart et al., supra; Maricq et al., supra; Kaplan and Horovitz, Proc. Natl. Acad. Sci. (USA) 90, 2227-2231, 1993; Troemel et al., Cell 83, 207-218,1995). Hence, genes required for ASH sensory responses were tested for their ability to perturb glutamate signaling.

[0064] We screened 11,000 mutagenized haploid genomes for animals that failed to respond to nose touch. Mutants isolated were subjected to a series of secondary screens, including dye-filling of the amphid sensory neurons, and responsiveness to osmotic shock and volatile repellents. Seven alleles of eat-4 were isolated in this screen, all of which were normal for dye-filling but were defective for all three ASH sensory behaviors. The amino acid sequence of the EAT-4 is shown on FIG. 3. ASH-mediated sensory responses to nose touch, osmotic shock, and volatile repellents were compared in wild type and eat-4, as has been previously described (Hart et al., supra; Maricq et al, supra; Kaplan and Horovitz, supra; Troemel et al., supra). Briefly, for nose touch, animals were tested 10 times each with a positive response being scored when animals either halted forward movement or initiated backward movement following the stimulus. For osmotic avoidance, 50-60 animals were placed in 1 cm rings formed with 8 M glycerol, and the number of animals that escaped the ring after 9 minutes were counted. For volatile avoidance, an eyelash was dipped in 1-octanol and held near an animal's nose, and responses were quantitated by recording the length of time that elapsed before the animals reversed locomotion.

[0065] All seven eat-4 strains isolated had similar behavioral defects. In particular, as is portrayed in Table 2, eat-4 strains had severe defects in the ASH-mediated touch, osmosensory, and volatile repellent responses. TABLE 2 Role of eat-4 in ASH sensory responses Osmotic Volatile Nose Touch Avoidance Avoidance Genotype: (% Respond) (% Escape) (seconds) wild type 86 +/− 3  2 +/− 1 2.9 +/− 0.9 eat-4(ky5) -  1 +/− 1 75 +/− 6 9.9 +/− 1.6 eat-4(n2474)  2 +/− 1 54 +/− 6 9.6 +/− 1.5

[0066] Errors indicate standard error of the mean in all cases. The number of animals and trials for each genotype were as follows: for nose touch, 10 animals and 100 trials; for osmotic avoidance, 60 animals and 5 trials; and for volatile avoidance, 25 animals and 25 trials.

[0067] Eat-4 Mutations Reduced Gα_(s)-induced Cytotoxicity but not Cell Swelling

[0068] The eat-4 gene was initially identified in screens for mutations that disrupted eating behavior (Avery, Genetics 133: 897-917, 1993). The eat-4 eating defect was caused by elimination of a glutamate-induced inhibitory synaptic signal (mediated by the M3 motor neuron), which could be observed in extracellular recordings of pharyngeal muscle activity (Raizen et al., Neuron 12, 483-495, 1994). Given the results described herein, the eating defects and the ASH sensory defects could both be explained by an underlying defect in glutamate signaling.

[0069] To investigate this possibility, neurodegenerative phenotypes were examined as described above. In these experiments, eat-4 mutations were found to be neuroprotective. The mutations significantly reduced Gα_(s)-induced cytotoxicity but had no apparent effect on cell swelling, as indicated in FIG. 2. In addition to reducing cytotoxicity, the eat-4 mutations also dramatically improved the locomotion rate of α_(s)(gf) animals. These results suggested that Gα_(s) neurotoxicity was at least partially mediated by endogenous glutamate signaling.

[0070] Apoptosis was not Required for Gα_(s) Neurotoxicity

[0071] Apoptosis is a naturally occurring process thought to play a critical role in the developing animal and is characterized morphologically by condensation of the chromatin followed by shrinkage of the cell body. Biochemically, DNA laddering, the degradation of nuclear DNA into oligonucleosomal fragments, is the hallmark of apoptosis. DNA laddering precedes cell death. Apoptosis is most likely dependent upon the activation of a cell death pathway. The best defined genetic pathway of cell death is in C. elegans where both effector (ced-3 and ced-4) and repressor (ced-9) genes have been isolated. Similar genes have been identified in mammals. Whether excitotoxic death occurs by apoptosis or by necrosis has remained controversial. This uncertainty is primarily due to lack of genetic control of the apoptosis pathway in the previously described models for excitotoxicity.

[0072] In our experiments, we found that a mutation in the ced-3 gene, which encodes an ICE protease and is required for apoptosis (Ellis and Horovitz, Cell 44: 817-829, 1986; Yuan et al., Cell 75: 641-652, 1993), had no effect on Gα_(s)-induced swelling or killing (see FIG. 2). Thus, apoptosis was not required for Gα_(s)-induced killing. However, in α_(s)(gf);unc-18 double mutants, a significant fraction of PVC neurons had a highly condensed morphology, and these PVC neuron corpses appeared to be engulfed by surrounding hypodermal cells, both of which are characteristic of apoptotic deaths (Ellis et al., Ann. Rev. Cell Biol. 7: 663-698, 1991). As shown in FIGS. 9A and 9B, in unc-18 L1 larvae, 13% of the PVC neurons exhibit the condensed morphologies characteristic of programmed cell deaths, which was apparent in both bright field (FIG. 9A) and fluorescence (FIG. 9B) optics. In unc-18 adults, 25% of the PVC neurons exhibited condensed morphologies and appeared to have been engulfed by surrounding hypodermal cells in the tail, as shown in FIG. 10A (bright field optics) and FIG. 10B (fluorescence optics). (Note that the position of the indicated cell body in FIG. 10B is much further posterior than in FIG. 7B). Gα_(s) neurotoxicity was concluded to be, in part, mediated by synaptic input to the dying cells, since unc-18 mutations impair synaptic vesicle exocytosis (Gengyo-Ando, et al., Neuron 11: 703-711, 1993; Hata et al., Nature 366: 347-351, 1993). Furthermore, these results suggest that Gα_(s) neurotoxicity occurs via two independent mechanisms. Synaptic input promotes an excitotoxic pattern of cell deaths; however, when synaptic input is impaired an apoptotic pattern emerges.

[0073] Screens for Compounds that Inhibit cAMP-based Neurodegeneration

[0074] As described herein, constitutive activation of the GTP-binding protein Gα_(s) induces a neurodegeneration phenotype that shares several properties with excitotoxic neuron loss in mammals. First, neuron loss occurs in two phases, whereby affected cells undergo a swelling response in young larvae, and subsequently die sometime during larval development. Second, neurons differ greatly in their susceptibility to Gα_(s)-induced toxicity, ranging from 0-88% of cells affected. Third, a mutation that impairs the function of voltage-dependent calcium channels and one that reduces glutamate neurotransmission are neuroprotective.

[0075] The acy-1 gene was identified in a screen for mutations that blocked G_(s)-induced killing and has been positionally cloned. The predicted ACY-1 protein (SEQ ID NO: 2) is highly similar (40% identical) to a mammalian adenylyl cyclase. Most consistent with this result is that Gα_(s)-induced neurotoxicity is mediated by changes in cyclic adenosine monophosphate (cAMP) levels. Mutations that prevent programmed cell death, also known as apoptosis, do not prevent Gα_(s)-induced neurotoxicity; however, when synaptic transmission was impaired (by an unc-18 mutation), a subset of the deaths appear to become apoptotic. These experiments suggested that excitotoxicity normally occurs by both apoptosis and a second cytotoxic pathway. Given these results, screens for compounds that inhibit cAMP signaling may be carried out to identify drugs that alter cAMP-based neurodegeneration and to provide therapies to ameliorate these disorders in humans and other mammals. These assays may be carried out in vivo or in vitro, and a number of exemplary assays now follow.

[0076] a) C. elegans assays

[0077] The microscopic nematode, C. elegans, is a useful model for studying neurodegeneration because it allows researchers to observe changes in neuronal cells within the living organisms over the three days required for a C. elegans to develop from a single cell zygote to a mature adult. The biology of the C. elegans nervous system, which includes 302 neurons, has been well documented. Furthermore, there are several similarities between the C. elegans and human nervous systems. For example, many of the C. elegans neurotransmitters are the same as human neurotransmitters. In addition, many C. elegans genes used both inside and outside of the nervous system have counterparts in mammals.

[0078] To identify a compound capable of inhibiting cAMP-based neurotoxicity, candidate compounds are screened for an ability to alter cAMP levels using a C. elegans strain carrying a reporter transgene operably linked to a promoter of a gene that is either (i) regulated by cAMP or (ii) involved in cAMP regulation. Exemplary promoters include the acy-1, unc-36, and eat-4 promoters. Other desirable promoters include any promoter from a nematode glutamate receptor (GluR) gene; such genes are listed, for example, in Table 3. TABLE 3 C. elegans glutamate signalling genes Gene Product Genetic locus iGluRs: C06E1.4 glr-1 CO6A8.8 BO280.12 F41B4.4 C43H6 K10D3.1 ZC196.c GluCls: GluCLα1 GluCLβ1 ZC317.3 T10G3 mGluRs: ZC506.4 mgr-1 F45H11

[0079] Once constructed, a transgenic C. elegans strain carrying such a reporter gene is treated with a candidate compound, or any number of compounds in combination, and animals are screened for alterations in cAMP levels as reflected by alterations in the levels of reporter gene expression.

[0080] Useful reporter genes are those whose expression is detectable, preferably, using simple and rapid techniques. Preferable reporter genes include, without limitation, green fluorescent protein (gfp), spectrally shifted green fluorescent proteins (Rizzuto et al., Curr. Biol. 6:183-188, 1996; Heim and Tsien, Curr. Biol. 6:178-182, 1996); lacZ, her-1(Perry et al., Gen. and Dev. 7(2): 216-228, 1993), and mec-4 (dominant) (Maricq et al., supra). Expression levels of these reporter genes may be directly measured by a variety of techniques known in the art. For example, if the reporter protein is a toxin (e.g., MEC-4), the expression level may be detected by measuring or observing cell viability. The expression level of a reporter protein with enzymatic activity (e.g., lacZ) may be quantitated using calorimetric substrates (e.g., 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal)). And reporter gene products such as GFP may be screened directly by visual inspection.

[0081] If desired, reporter proteins may be fusion proteins that incorporate portions of the sequences involved in cAMP regulation, for example, the ACY-1, UNC-36, or EAT-4 sequences. These fusion proteins are generated using nucleotide sequences and methods known in the art and described herein.

[0082] In one particular embodiment, such compound screens are carried out using rapid, high through-put assays. For example, transgenic C. elegans animals carrying acy-1::gfp reporter constructs are utilized. The animals are distributed into 96-well microtiter dishes such that there is one animal per well. Candidate compounds are then individually or combinatorially added to the wells and assessed for an ability to reduce GFP expression as a means to test for an ability to inhibit cAMP-based neurodegeneration. GFP assays may be carried out by any means, but are preferably monitored using a microtiter plate fluorescence reader.

[0083] In an alternative compound screen, the reporter protein need not be GFP. For example, the transgenic animal may carry a lacZ reporter gene and be distributed into microtiter wells as described above. Following compound administration, transgenic animals are subjected to standard β-galactosidase activity assays described in the art (see, for example, Ausubel et al., supra). The Promega β-gactosidase enzyme assay system with reporter gene lysis buffer kit (Catalog # E2000) may be employed in this rapid high throughput 96 well assay system. By this method, reporter lysis buffer is added to each well. The C. elegans extracts are then incubated with the buffer and the o-nitrophenyl-β-D-glactopyranoside (OPTG) substrate provided in the kit. Optical density of the plate is then measured on a microtiter plate reader. Again, a reduced level of lacZ activity in a compound-treated well as compared to an untreated well indicates that the compound has an ability to inhibit cAMP-based neurodegeneration.

[0084] In addition, a variety of methods may be used in combination to screen for compounds capable of inhibiting cAMP-based neurodegeneration. For example, a C. elegans carrying two different expression constructs (e.g., the acy-1 promoter operably linked to gfp and the glr-1 promoter operably linked to lacZ) may be used to screen for a compound capable of inhibiting cAMP-based neurodegeneration by assaying for a reduction in the expression of both the acy-1 and eat-4 genes. In this assay, preferred compounds are capable of reducing the expression levels of both GFP and lacZ. However, a decrease in expression of one reporter gene (e.g., gfp), but not the other reporter gene (e.g., lacZ) identifies compounds capable of targeting particular components in a neurodegenerative pathway (in this case, the acy-1 gene). Such compounds may be useful for treating particular types of neurodegenerative disorders.

[0085] In addition, nematode screens for compounds capable of inhibiting cAMP-based neurodegeneration may be based upon both neuroprotection and reporter gene expression. By this approach, for example, a transgenic glr-1::α_(s)(gf) C. elegans is transformed with a second worm marker (e.g., the acy-1::gfp expression vector). Compound-treated glr-1::α_(s)(gf). acy-1::gfp double transgenic animals are then screened, for example, for improved locomotion (i.e., a compound affecting the glr-1 gene), reduction of GFP expression (i.e., a compound affecting the acy-1 gene), or both (i.e., a compound affecting both the glr-1 and acy-1 genes) as compared to untreated glr-1::α_(s)(gf); acy-1::gfp double trangenic animals. Again, gene-specific compounds may be useful for treating neurodegenerative disorders involving specific genes.

[0086] In yet another approach, compounds which affect neurodegenerative signals generated by a mammalian glutamate receptor may also be employed in a C. elegans screen. A large number of mammalian glutamate receptors (GluRs) have been previously described, and a comprehensive list of these proteins may be found in Hollmann and Heinemann (Ann. Rev. Neurosci. 17: 31-108, 1994). To carry out such a screen, the coding regions of one or more of these genes are inserted into a C. elegans expression vector such that the expression of the gene product is directed by the glr-1 gene promoter. This construct is microinjected into a glr::α_(s)(gf) transgenic C. elegans. Since only a subset of the glr-1 expressing neurons die in such animals, any additional cell death (as measured, for example, by increased paralysis, neuronal swelling, or neurodegeneration) may be attributed to mammalian GluR expression. Candidate compounds are then administered to these animals, and differences observed in compound-treated animals versus untreated animals are used to identify a compound having an ability to affect mammalian GluR signalling. Again, compounds identified by this assay are useful for treating neurodegenerative disorders in a mammal.

[0087] b) Mammalian Cell Assays

[0088] Mammalian cells carrying a reporter gene operably linked to the promoter of a gene either regulated by cAMP or involved in cAMP regulation, for example, the mammalian homologues of the acy-1, unc-36, or eat-4 genes, may also be used to screen for compounds that inhibit cAMP-based neurodegeneration. In one particular example, the promoter of the murine adenylyl cyclase type 9 encoding gene may be used to direct the expression of a reporter (e.g., GFP) in a mammalian expression vector. This vector is transfected into a mammalian cell by any of a number of different transfection methods well known in the art (e.g., electroporation, CaPO₄ precipitation, or DEAE-Dextran). Preferably, the mammalian cell is a mouse neuronal cell line, for example, a PC12 cell line. Candidate compounds are added to the culture medium of the transfected cells, and the level of expression of the reporter gene is measured and compared to a control, untreated cell line. A reduced level of reporter gene expression in a compound-treated cell line indicates that the compound has an ability to inhibit cAMP-based neurodegeneration in mammalian cells.

[0089] In addition, such a mammalian cell line may be transfected with more than one reporter gene operably linked to more than one cAMP regulatory gene promoter. For example, a mammalian cell transfected with a gfp-adenylyl cyclase type 9 construct may be doubly transfected with a construct comprising a mammalian unc-36 promoter operably linked to a second reporter (e.g., luciferase). Following addition of candidate compounds to the culture medium of doubly transfected cells, GFP expression is analyzed, for example, by flow cytometric analysis of half of the compound-treated cell population, and the remaining half is assayed for luciferase activity using known methods (e.g., the luciferase assay kit commercially available from Promega). By comparing GFP and luciferase expression levels to those in untreated cells, a compound capable of altering cAMP-based neurodegeneration is identified. Furthermore, a compound capable of affecting, for example, the murine adenylyl cyclase type 9 gene but not the mammalian unc-36 gene may also be isolated. Such a compound may be useful for treating specific types of neurodegenerative disorders in mammals.

[0090] Alternatively, mammalian cells which endogenously express homologues of C. elegans genes involved in cAMP regulation or regulated by cAMP may be used to identify compounds capable of altering cAMP-induced neurodegeneration. According to this method, following administration of a candidate compound, endogenous gene expression is measured by any of a variety of nucleic acid or immunological based assays including, without limitation, Northern blot, Western blot, and ELISA analyses. Compounds affecting endogenous gene expression levels as compared to untreated cells are useful for treating cAMP-based neurodegeneration.

[0091] c) Animal Models

[0092] A number of animal models exist for the study of neurodegenerative disorders and find use in the screening methods described herein. For example, such models may serve as a system in which to screen candidate compounds being tested de novo for an ability to alter cAMP-based neurodegeneration or as a secondary screen for testing compounds isolated in a C. elegans, yeast, or mammalian cell culture assay (for example, those assays described herein). Candidate compounds may be administered to animals prior to neurological damage to assay for an ability to prevent cAMP-based neurodegeneration. Alternatively, candidate compounds may be assessed for an ability to treat cAMP-based neurodegeneration following neurological insult. Animal models may also serve to determine the dosage requirement for an effective compound.

[0093] Particularly useful animal models include, without limitation, Parkinson's disease (PD) rat models, which are established by injecting the catecholamine-specific neurotoxin, 6-hydroxydopamine (6-OHDA), into the medial forebrain bundle or the substantia nigra pars compact to achieve a rapid degeneration of the nigrostriatal pathway, or into the striatum to achieve progressive degeneration, as has been described (see, for example Gerlach and Riederer, J. Neural. Transm. 103 (8-9): 987-1041, 1996; Bernard et al., J. Comp. Neurol. 368 (4): 553-568, 1996; Asada et al., Ex. Neurol. 139 (2): 173-187, 1996). Alternatively, rats may be rendered “epileptic” (i.e., induced to suffer brain seizures which often result in neuronal cell death) by administration of a variety of compounds including, for example, intravenous injection of bicuculline (Blennow et al., J. Cereb. Blood Flow Metab. 5: 439-445, 1995) or daily application of low intensity electrical stimulation. Finally, neuronal cell death which often results from stroke-induced ischemia may be mimicked by the 4-vessel occlusion experimental model described by Pulsinelli et al. (Ann. Neurol. 11: 491-498, 1982) and Francis and Pulsinelli (Brain Res. 243: 271-278, 1982).

[0094] d) Candidate inhibitors of cAMP-based neurodegeneration

[0095] A number of compounds have been shown to affect cAMP levels, and these provide good candidates for inhibitors of neurodegeneration. Such compounds are commercially available (e.g., from Research Biochemicals International) and include, without limitation, agonists of receptors that couple to Gi and inhibit adenylyl cyclases. Alpha 2 adrenergic receptor agonists (including B-HT 920 diHCl and Xylazine HCl), opioid delta receptor agonists (including [D-Ala2, D-Leu5]-enkephalin and [D-Pen2,5]-enkephalin) and D2 dopamine receptor agonists (including bromocriptine methane sulfonate and Quinelorane 2HCl) all inhibit adenylyl cylcases and may be assessed in screens described herein for an ability to inhibit cAMP-based neurodegeneration.

[0096] Therapeutics for Treating Human Neurodegenerative Disorders

[0097] A number of human neurological disorders are characterized by a loss of neurons through a degenerative process. Compounds isolated as described above based on their effect on cAMP levels are useful in treating these disorders. In addition, drugs known to lower cAMP levels are also useful therapeutics for treating, preventing, or slowing neurodegeneration. In particular, disorders that may be treated using such compounds include, without limitation, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, multiple sclerosis, epilepsy, and stroke.

[0098] Compounds that alter cAMP levels may be administered by any appropriate route. For example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, by suppositories, or by oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

[0099] Methods well known in the art for making formulations are found, for example, in “Remington's Pharmaceutical Sciences.” Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

[0100] Dosage is determined by standard techniques and is dependent, for example, upon the weight of the mammal and the type or extent of disorder being treated.

[0101] Diagnostics for Neurodegenerative Disorders

[0102] To determine whether an individual either has or is likely to develop a neurodegenerative disorder, that individual is screened for mutations in genes which are either involved in regulation of cAMP, or are regulated by cAMP, for example, genes encoding the adenylyl cyclases, G proteins, or human homologues of UNC-36 or EAT-4 proteins described herein. Such assays may be carried out by any standard technique including, without limitation, methods involving sequencing or mismatch binding or cleaving assays. In one particular example, a nucleic acid sample derived from the neuronal cells of an individual may be isolated (for example, by PCR amplification), and a cAMP regulatory gene (or a portion thereof) subjected to rapid sequence analysis by automated sequencing techniques using primers generated from sequences described herein and in the art.

[0103] Alternatively, an individual who either has or is likely to develop a neurodegenerative disorder may be screened for altered expression of adenylyl cyclases, G proteins, or the human homologues of UNC-36 or EAT-4 proteins, or for an increased level of cellular cAMP, particularly in neuronal cells. Such assays may be carried out, for example, using any standard nucleic acid-based assay (e.g., Northern blot analysis) or immunological assay (e.g., enzyme-linked immunosorbent assay (ELISA)), preferably in a high through-put assay format. In one particular example, neuronal cells obtained from an individual being screened for a neurodegenerative disorder may be isolated and analyzed for the expression of adenylyl cyclases, G proteins, and the human homologues of UNC-36 and EAT-4 proteins by ELISAs using fluorophore-tagged antibodies directed toward these proteins as probes. Individuals incapable of expressing certain proteins may be identified by rapidly assessing the results of these ELISAs in a microtiter plate format.

[0104] In particular examples, candidate human genes, for example, those involved in cAMP regulation, are examined for genetic linkage to hereditable forms of neurodegeneration found in humans or, as a model system, mice. These genetic linkages are assessed using standard methods known in the art, and, upon identification of a linkage with neurodegeneration, diagnostic mutation detection is conducted as described herein. Listed in Table 4 are exemplary candidate human genes likely involved in neurodegeneration. TABLE 4 Candidate Human Neurodegeneration Genes Class of Protein Gene Product Phosphodiesterases PDE4A PDE4B PDE6G PDE7A G alpha subunits GNAS1 GNAI1 GNAI2 GNAI3 Golf Protein Phosphatases PPP1CB PPP1CC PPP2CA PPP2R4 PPP2R5A PPP2R5C PPP2R5D PPP2R5E PPP3CA PPP3CB PPP3R1

[0105] Methods for Isolating Genes Involved in Neurodegeneration

[0106] a) C. elegans Screens

[0107] Additional genes involved in neurodegeneration may be isolated using the methods described herein. For example, a gene involved in neurodegeneration may be isolated by inducing paralysis and neurodegeneration in C. elegans. This is accomplished, for example, by generating a nematode strain carrying a constitutively active (GTP-ase defective) Gα_(s) subunit gene operably linked to a glutamate receptor (GluR) promoter, such as glr-1. The transgenic C. elegans is screened for gene mutations which restore locomotion and reduce neurodegeneracy (cytotoxicity genes) or which reduce Gα_(s)-induced neuronal cell swelling (swelling genes).

[0108] I. Gα_(s)-associated Cytotoxicity Genes

[0109] To isolate a Gα_(s)-associated cytotoxicity gene, glr-1::α_(s)(gf) transgenic nematodes are mutagenized, for example, with EMS or γ-irradiation, and then screened for mutants with both improved locomotion and increased survival of the Gα_(s) expressing neurons. If desired, these mutants may be genetically mapped and placed into complementation groups. The genes identified in these mutants may then be positionally cloned.

[0110] II. Gα_(s)-associated Swelling Genes

[0111] To isolate a Gα_(s)-associated swelling gene, glr-1::α_(s)(gf) transgenic nematodes are mutagenized, for example, with EMS or γ-irradiation. First stage larvae are then isolated and screened by fluorescence microscopy (as described herein) for mutants which show a reduced incidence of swelling of Gα_(s)-expressing neurons. These mutants may be genetically mapped and positionally cloned.

[0112] III. Other Genes

[0113] The transgenic animals developed to identify compounds that inhibit cAMP-based neurodegeneration may also be used to identify additional genes involved in neurodegeneration. For example, C. elegans doubly transgenic for acy-1::gfp; glr-1::α_(s)(gf) may be mutagenized (for example, with EMS or γ-irradiation) and then analyzed for restored locomotion and reduced neurodegeneration (i.e., for a mutation in a gene which affects the glr-1 promoter) or a reduced level of GFP expression (i.e., for a mutation in a gene which affects the acy-1 promoter), or both (i.e., a mutation in a gene which affects both acy-1 and glr-1 gene promoters).

[0114] In an alternative approach, physiological stresses, such as ischemia, due to, for example, the interruption of available oxygen, may be administered to EMS or γ-irradiated worms to induce neurodegeneration. Mutants which resist ischemia-induced neurodegeneration may then be isolated and characterized to identify the neuroprotective mutant gene.

[0115] A gene involved in neurodegeneration may be cloned and sequenced by standard methods (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 and Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989). If desired, a protein product from this gene may then be produced, for example, by inserting the cloned gene into an expression vector and introducing this vector into bacterial or eukaryotic cells to produce recombinant proteins. Techniques for such manipulations are disclosed in Sambrook et al., supra, and are well known in the art. Genes involved in neurodegeneration or their protein products may be used in any of the screening or diagnostic assays described herein.

[0116] b) Yeast Screens

[0117] Another approach to identify genes involved in neurodegeneration utilizes yeast carrying a reporter gene operably linked to a promoter from a cAMP regulatory gene, and, preferably, a mammalian cAMP regulatory gene. The reporter construct is stably introduced into yeast by any standard method. A cDNA library (preferably, from a mammalian cell) is then introduced into the yeast carrying the reporter construct, and yeast colonies exhibiting an increased level of reporter gene expression (e.g., lacZ reporter yeast with increased blue colony color on X-Gal) are identified. Such yeast carry a cDNA capable of binding to the cAMP promoter and are therefor good candidates for a gene involved in cAMP-based neurodegeneration. If desired, the promoter sequences from the newly isolated gene may also be used to generate reporter cells (e.g., reporter yeast or transgenic C. elegans ) to identify additional genes involved in cAMP-based neurodegeneration.

[0118] Moreover, this yeast system may be used to screen for compounds which inhibit the ability of the cDNA to induce reporter gene expression. Such compounds provide good candidates for therapeutics for treating cAMP-based neurodegeneration.

[0119] Mammalian Genes Involved in Neurodegeneration

[0120] a) Mammalian eat-4 genes

[0121] Any of a variety of procedures well known in the art may be utilized to clone the mammalian homologues of the nematode eat-4 gene, and one so skilled will routinely adapt one of these methods in order to obtain the desired gene.

[0122] One such method for obtaining a mammalian gene sequence is to use an oligonucleotide probe generated by the C. elegans eat-4 gene sequence to screen a mammalian cDNA or genomic DNA library for sequences which hybridize to the probe. Hybridization techniques are well known to the skilled artisan, and are described, for example, in Ausubel et al., supra, and Sambrook et al., supra. cDNA or genomic DNA library preparation is also well known in the art. A large number of prepared nucleic acid libraries are also commercially available. The oligonucleotide probes are readily designed using the sequences described herein and standard techniques. The oligonucleotide probes may be based upon the sequence of either strand of DNA encoding the eat-4 gene product (SEQ ID NO: 1). Exemplary oligonucleotide probes are degenerate probes (i.e., a mixture of all possible coding sequences for the EAT-4 protein).

[0123] If desired, the cloned gene may be inserted into an expression vector and introduced into bacterial or eukaryotic cells to produce the mammalian EAT-4 protein. Techniques for such manipulations are disclosed, for example, in Sambrook et al., supra. The mammalian eat-4 gene or gene product may be used in the neurodegeneration screening or diagnostic assays described herein.

[0124] b) eat-4 related C. elegans genes

[0125] Genes related to eat-4 may be isolated by methods similar to those described above. For example, a cosmid library from C. elegans may be screened with the degenerate oligonucleotide probes described above under low stringency hybridization conditions to isolate eat-4 related C. elegans genes. Oligonucleotide probes may be prepared from these gene sequences and may be used to screen mammalian nucleic acid libraries for hybridizing sequences, thus, identifying mammalian homologues of these eat-4 related genes.

[0126] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0127] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the appended claims.

[0128] Other embodiments are within the claims. 

What is claimed is:
 1. A method for identifying a compound to treat or prevent the onset of a neurodegenerative disorder, said method comprising the steps of: a) providing a cell comprising a reporter gene operably linked to a cAMP regulatory gene or promoter; b) contacting said cell with a candidate compound; and c) measuring expression of said reporter gene, a change in said expression in response to said candidate compound identifying a compound that is useful to treat or prevent the onset of a neurodegenerative disorder.
 2. The method of claim 1, wherein said cAMP regulatory gene is an acy-1 gene.
 3. The method of claim 1, wherein said cAMP regulatory gene is an eat-4 gene.
 4. The method of claim 1, wherein said cAMP regulatory gene is an unc-36 gene.
 5. The method of claim 1, wherein said cAMP regulatory gene is a glutamate receptor.
 6. The method of claim 1, wherein said change in said expression is a decrease in expression.
 7. The method of claim 1, wherein said cell is present in an animal.
 8. The method of claim 7, wherein said animal is a nematode.
 9. The method of claim 8, wherein said nematode is C. elegans.
 10. The method of claim 7, wherein said animal is a mammal.
 11. The method of claim 10, wherein said mammal is a rodent.
 12. The method of claim 1, wherein said neurodegenerative disorder is Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, stroke, or epilepsy.
 13. A cell for identifying a compound to treat or prevent the onset of a neurodegenerative disorder, said cell comprising a reporter gene operably linked to a promoter of a cAMP regulatory gene.
 14. The cell of claim 13, wherein said cell is present in an animal.
 15. The cell of claim 14, wherein said animal is C. elegans.
 16. The cell of claim 14, wherein said animal is a rodent.
 17. The cell of claim 13, wherein said neurodegenerative disorder is Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, stroke, or epilepsy.
 18. A method for treating or preventing the onset of a neurodegenerative disorder in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a compound that decreases a neuronal cAMP level.
 19. The method of claim 18, wherein said mammal is a human.
 20. The method of claim 19, wherein said neurodegenerative disorder is Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, stroke, or epilepsy.
 21. A method for identifying a mammal having or likely to develop a neurodegenerative disorder, said method comprising determining whether said mammal has an increased level of cellular cAMP in a neuron, said increased level indicating that said mammal has or is likely to develop a neurodegenerative disorder.
 22. A method for identifying a mammal having or likely to develop a neurodegenerative disorder, said method comprising determining whether said mammal has a mutation in a cAMP regulatory gene, said mutation being an indication that said mammal has or is likely to develop a neurodegenerative disorder.
 23. The method of claim 22, wherein said mutation is in an adenylyl cyclase gene.
 24. The method of claim 23, wherein said adenylyl cyclase gene is the acy-1 gene.
 25. The method of claim 22, wherein said mutation is in the unc-36 gene.
 26. The method of claim 22, wherein said mutation is in the eat-4 gene.
 27. The method of claim 22, wherein said mutation is in a gene encoding a Gα_(s) subunit.
 28. The method of claim 22, wherein said mutation results in an increase in a neuronal cAMP level.
 29. The method of claim 21 or 22, wherein said mammal is a human.
 30. The method of claim 29, wherein said neurodegenerative disorder is Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, stroke, or epilepsy.
 31. A method for identifying a gene involved in neurodegeneration, said method comprising the steps of: a) providing a nematode comprising an expression construct, said expression construct comprising a promoter derived from a cAMP regulatory gene operably linked to a reporter gene; b) isolating a mutant of said nematode exhibiting an altered level of reporter gene expression; and c) identifying said gene comprising said mutation, said gene being involved in neurodegeneration.
 32. A method for identifying a gene involved in neurodegeneration, said method comprising the steps of: a) providing a nematode comprising a glutamate receptor (GluR) promoter operably linked to a gene encoding a GTP-ase defective Gα_(s) subunit; b) isolating a mutant of said nematode exhibiting a decreased level of paralysis and neurodegeneration; and c) identifying said gene comprising said mutation, said gene being involved in neurodegeneration.
 33. The method of claim 31 and 32, wherein said nematode is C. elegans.
 34. A method for identifying a gene involved in neurodegeneration, said method comprising the steps of: (a) providing a cell comprising a cAMP regulatory gene promoter operably linked to a reporter gene; (b) introducing into said cell a candidate gene capable of expressing a candidate protein; and (c) measuring reporter gene expression in said cell, an increase in said reporter gene expression in the presence of said candidate protein indicating that said candidate gene is involved in neurodegeneration.
 35. The method of claim 34, wherein said cell is yeast.
 36. The method of claim 34, wherein said cAMP regulatory gene is an acy-1 gene.
 37. The method of claim 34, wherein said cAMP regulatory gene is an eat-4 gene.
 38. The method of claim 34 wherein said cAMP regulatory gene is an unc-36 gene.
 39. The method of claim 34, wherein said cAMP regulatory gene is a glutamate receptor-encoding gene.
 40. A mammalian EAT-4 polypeptide.
 41. A purified nucleic acid encoding the polypeptide of claim
 40. 42. The nucleic acid of claim 41, wherein said mammal is a human.
 43. A vector comprising the nucleic acid of claim 41 said vector being capable of directing expression of the said polypeptide in a vector-containing cell.
 44. A cell that contains the nucleic acid of claim
 41. 